IL-2-mediated hepatotoxicity: knowledge gap identification based on the irAOP concept.

Drug-induced hepatotoxicity constitutes a major reason for non-approval and post-marketing withdrawal of pharmaceuticals. In many cases, preclinical models lack predictive capacity for hepatic damage in humans. A vital concern is the integration of immune system effects in preclinical safety assessment. The immune-related Adverse Outcome Pathway (irAOP) approach, which is applied within the Immune Safety Avatar (imSAVAR) consortium, presents a novel method to understand and predict immune-mediated adverse events elicited by pharmaceuticals and thus targets this issue. It aims to dissect the molecular mechanisms involved and identify key players in drug-induced side effects. As irAOPs are still in their infancy, there is a need for a model irAOP to validate the suitability of this tool. For this purpose, we developed a hepatotoxicity-based model irAOP for recombinant human IL-2 (aldesleukin). Besides producing durable therapeutic responses against renal cell carcinoma and metastatic melanoma, the boosted immune activation upon IL-2 treatment elicits liver damage. The availability of extensive data regarding IL-2 allows both the generation of a comprehensive putative irAOP and to validate the predictability of the irAOP with clinical data. Moreover, IL-2, as one of the first cancer immunotherapeutics on the market, is a blueprint for various biological and novel treatment regimens that are under investigation today. This review provides a guideline for further irAOP-directed research in immune-mediated hepatotoxicity.

Interleukin-2-induced skin inflammation.

Recombinant human IL-2 has been used to treat inflammatory diseases and cancer; however, side effects like skin rashes limit the use of this therapeutic. To identify key molecules and cells inducing this side effect, we characterized IL-2-induced cutaneous immune reactions and investigated the relevance of CD25 (IL-2 receptor α) in the process. We injected IL-2 intradermally into WT mice and observed increases in immune cell subsets in the skin with preferential increases in frequencies of IL-4- and IL-13-producing group 2 innate lymphoid cells and IL-17-producing dermal γδ T cells. This overall led to a shift toward type 2/type 17 immune responses. In addition, using a novel topical genetic deletion approach, we reduced CD25 on skin, specifically on all cutaneous cells, and found that IL-2-dependent effects were reduced, hinting that CD25 - at least partly - induces this skin inflammation. Reduction of CD25 specifically on skin Tregs further augmented IL-2-induced immune cell infiltration, hinting that CD25 on skin Tregs is crucial to restrain IL-2-induced inflammation. Overall, our data support that innate lymphoid immune cells are key cells inducing side effects during IL-2 therapy and underline the significance of CD25 in this process.

Targeted GATA3 knockdown in activated T cells via pulmonary siRNA delivery as novel therapy for allergic asthma.

GATA3 gene silencing in activated T cells displays a promising option to early-on undermine pathological pathways in the disease formation of allergic asthma. The central transcription factor of T helper 2 (Th2) cell cytokines IL-4, IL-5, and IL-13 plays a major role in immune and inflammatory cascades underlying asthmatic processes in the airways. Pulmonary delivery of small interfering RNAs (siRNA) to induce GATA3 knockdown within disease related T cells of asthmatic lungs via RNA interference (RNAi) presents an auspicious base to realize this strategy, however, still faces some major hurdles. Main obstacles for successful siRNA delivery in general comprise stability and targeting issues, while in addition the transfection of T cells presents a particularly challenging task itself. In previous studies, we have developed and advanced an eligible siRNA delivery system composed of polyethylenimine (PEI) as polycationic carrier, transferrin (Tf) as targeting ligand and melittin (Mel) as endosomolytic agent. Resulting Tf-Mel-PEI polyplexes exhibited ideal characteristics for targeted siRNA delivery to activated T cells and achieved efficient and sequence-specific gene knockdown in vitro. In this work, the therapeutic potential of this carrier system was evaluated in an optimized cellular model displaying the activated status of asthmatic T cells. Moreover, a suitable siRNA sequence combination was found for effective gene silencing of GATA3. To confirm the translatability of our findings, Tf-Mel-PEI polyplexes were additionally tested ex vivo in activated human precision-cut lung slices (PCLS). Here, the formulation showed a safe profile as well as successful delivery to the lung epithelium with 88% GATA3 silencing in lung explants. These findings support the feasibility of Tf-Mel-PEI as siRNA delivery system for targeted gene knockdown in activated T cells as a potential novel therapy for allergic asthma.

Low Energy Electron Irradiation Is a Potent Alternative to Gamma Irradiation for the Inactivation of (CAR-)NK-92 Cells in ATMP Manufacturing.

Background: With increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these "off-the-shelf" therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function. Methods: Effectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity assays, and comet assays, amongst others. Results: Our results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NK-mediated specific lysis of tumor cells was maintained at stable levels for three days post-irradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30. Conclusions: The presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application.

From Cancer to Immune-Mediated Diseases and Tolerance Induction: Lessons Learned From Immune Oncology and Classical Anti-cancer Treatment.

Success in cancer treatment over the last four decades has ranged from improvements in classical drug therapy to immune oncology. Anti-cancer drugs have also often proven beneficial for the treatment of inflammatory and autoimmune diseases. In this review, we report on challenging examples that bridge between treatment of cancer and immune-mediated diseases, addressing mechanisms and experimental models as well as clinical investigations. Patient-derived tumor xenograft (PDX) (humanized) mouse models represent useful tools for preclinical evaluation of new therapies and biomarker identification. However, new developments using human ex vivo approaches modeling cancer, for example in microfluidic human organs-on-chips, promise to identify key molecular, cellular and immunological features of human cancer progression in a fully human setting. Classical drugs which bridge the gap, for instance, include cytotoxic drugs, proteasome inhibitors, PI3K/mTOR inhibitors and metabolic inhibitors. Biologicals developed for cancer therapy have also shown efficacy in the treatment of autoimmune diseases. In immune oncology, redirected chimeric antigen receptor (CAR) T cells have achieved spectacular remissions in refractory B cell leukemia and lymphoma and are currently under development for tolerance induction using cell-based therapies such as CAR Tregs or NK cells. Finally, a brief outline will be given of the lessons learned from bridging cancer and autoimmune diseases as well as tolerance induction.

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α), and interleukin 1 alpha (IL-1α) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.